Water-soluble combination products of gossypol and proteins



Patented Jan. 8, 1 952 WATER-SOLUBLE COMBINATION PRODUCTS OF GOSSY POLAND PROTEINS Aaron M. Altschul and Leah E. Castillon, New rleans,.La.,assignors to the United, States of America. as represented by the.Secretary of Agriculture No Drawing. Application July 11,1950, SerialNo. 173,221

3. Claims.

(Granted under. the act of March 3, 1883, as

amended April 30, 1928; 370 0. G. 757).

This invention relates to the preparation of water soluble products ofgossypol by combination with materials of natural origin. Gossypol is anaturally occurring pigment of cottonseed which is present to the extentof 0.5 to 1.5 percent by weight of the kernels. This pigment isconcentrated in the pigment glands of the cottonseed and comprises 35 to50 percent of the weight of these glands. Certain solvents such aspetroleum naphthas or chlorinated hydrocarbons can be used to'extractthe oil from flaked cottonseed kernels without materially affecting thepigment glands or removing substantial quantities of their contents.Other solvents such as alcohols, ethers, and ketones rupture the pigmentglands and extract their contents with the oil present in the seed.

It has been found that oil-free cottonseed meals containing intactpigment glands are toxic when fed in certain specific amounts to certainanimals such as chicks and pigs. "When, however, the pigment glandcontents are removed along with the oil, the resulting meal gives noevidence of any physiological toxic effects. It has been establishedthat intact pigment glands of cottonseed are toxic to animals andconsiderable experimental evidence suggests that one of the principalfactors contributing to the toxicity of pigment glands is the gossypolcontained therein.

In the, normal practice of processing cottonseed for oil and meal by thehydraulicor continuous screw-press methods, in which heat and pressureare applied to flaked or rolled cottonseed meats, the pigment glands areruptured and their contents react with the surrounding extraglandularmaterials. Whereas in raw cottonseed meats, the amount of free gossypol,that is gossypol which is readily extractable by aqueous acetone orchloroform, varies from 0.5 to 1.5 percent, very little free gossypolcan be detected in hydraulic or screw-press meal. In some of the newlydeveloped methods of processing by solvent extraction of the oil, thecottonseed meats are cooked prior to extraction in order to facilitateextraction and the oil-free flakes are cooked after extraction todetoxify them. Gossypol is also destroyed by these processes andgenerally cannot be detected in the oil-free meals.

If, however, the oil is extracted withoutapplication of heats bysolvents which do not cause the rupture of the pigment glands or by thenew method of fractionation whereby the pigment glands are separatedintact from the oil and meal (pending application of Boatner, Hall, andMerrifield, Serial No. 675,118, filed June 7, 1946,

now U. S. Patent No. 2,482,141, issued September 20, 1947), the gossypolremains unchanged. By use of the solvent extraction method, the gossypolis found in the oil-free meal and in the gland fractionation process, inthe separated pigment glands. In either-case gossypol can be recoveredfrom the meal or pigment glands by extraction with a suitable solventsuch as aqueous acetone and can be purified to yield pure crystallinematerial.

Gossypol is a new byproduct of processing cotton seed and can materiallyincrease the value of cottonseed to the farmer and processor. It hasbeen suggested that gossypol may have pharmaceutical uses and may beused as an insecticide. Biological research with this material and itsapplication to new uses is difficult, however, because of itsinsolubility in water.

The object of our invention is to prepare products of gossypol which areeither water-soluble or water-dispersible. We have found that gossypolmay be combined with protein to form a new product which is soluble inneutral water or buffer solution to the extent of approximately 10percent. Whereas a mixture of gossypol and water is not toxic togoldfish, a solution of the new gossypol-protein product in Water in aconcentration of one part to 20,000 parts of water kills 'trated but notlimited by the following examples.

Example 1.-Ten grams of purified peanut protein was added to one literof distilled water. The mixture was treated with dilute aqueous sodiumhydroxide solution until the pH of the solution was increased to 8.5. Atthis pH value the protein was completely soluble. Ten grams of puregossypol was added to this protein solution and dilute aqueous sodiumhydroxide was added drop by drop until the pH of the solution wasincreased to 10.3 and the gossypol was solubilized. Immediatelythereafter an aqueous solution of hydrochloric acid was added dropwiseuntil the pH of the solution was reduced to 6.8 to 7.0. During theentire course of. the preparation the solution was constantly agitated.The solution was then frozen and lyophilized, i. e., the water wasremoved from the material in the frozen state under conditions of highvacuum, low pressure, and low temperature. A yield of 20 grams offluffy, yellow-colored, dry product was obtained. This material wassoluble in water at neutral pH.

Goldfish placed in a medium containing one part of this materialdissolved in 20,000 parts of water at neutral pH died within one hour.Goldfish placed in a medium containing one part of pure gossypolsuspended in 1,000 parts of water at neutral pH did not .die in fivedays.

Example 2.Two-tenths of a gram of peanut protein was added to one literof distilled water and the mixture was stirred while dilute aqueousalkali was added to dissolve the protein. To this solution was added twograms of pure gossypol and enough aqueous alkali to increase the pH ofthe solution to 10.3. When complete solution of both the protein andgossypol was attained, the solution was neutralized by the addition ofacid under conditions of constant stirring. The neutral solution wasfrozen and dried by lyophilization and a yellow-colored product weighing2.18 grams was recovered. This material was soluble in water at neutralpH and was lethal to goldfish in a concentration of 1 to 20,000 ofwater.

Example 3.One gram of gossypol was added to a liter of one-tenth percentsolution of peanut protein at pH of 8.5. By addition of more aqueoussodium hydroxide solution, the pH of the solution was increased to10.3,thereby making the gossypol completely soluble. While the solution wasconstantly stirred, aqueous hydrochloric acid solution was added untilthe pH of the solution was lowered to 4.5, whereupon a yellow flocculentprecipitate formed. The mixture was centrifuged, and the colorlessaqueous supernatant solution was decanted. Fifty milliliters ofdistilled water was used to transfer the precipitated gossypol-proteinproduct from centrifuge bottles into a large flask in which it wasfrozen and dried by lyophilization. This material was soluble in waterat neutral pH and was lethal to goldfish in a dilution of 1 part to20,000 parts of water.

Example 4.Ten grams of casein was added to one liter distilled water.Aqueous sodium hydroxide solution was added dropwise until the caseinwas entirely dissolved. To this solution there was added grams of puregossypol and sufficient aqueous sodium hydroxide solution to increasethe pH of the solution to approximately 11.0. All of the gossypol wentinto solution after which the pH of the solution was lowered to 7.0 bythe addition of aqueous hydrochloric acid solution, and it was frozenand dried by lyophilization. This dried product was soluble in water atneutral pH and was lethal to goldfish.

The invention herein described may be manufactured and used by or forthe Government of the United States of America for governmental purposesthroughout the world, without the payment to us of any royalty thereon.

Having thus described our invention, what we claim is:

1. A process of preparing a water-soluble, toxic, gossypol productcomprising dissolving gossypol and protein in dilute aq'ueous alkali,and neutralizing with acid.

2. A process of preparing a water-soluble tqxic gossypol productcomprising dissolving gossypol and protein in dilute aqueous alkali,neutralizin with acid, and drying by lyophilization.

3. The process of claim 1 in which the protein is peanut protein.

4. The process of claim 1 in which the protein is casein.

5. A process of preparing a water-soluble toxic gossypol productcomprising dissolving protein in dilute aqueous sodium hydroxide, addinggossypol to the solution, and dissolving it by adding dilute aqueoussodium hydroxide to about pH 10.3, adding hydrochloric acid to reducethe pH to the range 6.8 to 7.0, freezing the solution and lyophilizingto obtain a dry product. 7 4,

6. The process of claim 5 in which the protein is peanut protein.

'7. The water-soluble toxic gossypol product produced by the process ofclaim 2 in which the protein is peanut protein.

8. The water-soluble toxic gossypol product produced by the process ofclaim 2 in which the protein is casein protein. v,

9. The water-soluble, toxic gossypol product produced by the process ofclaim 2 in which the protein is taken from the class consisting of pea.nut and casein protein.

AARON M. ALTSCHUL. LEAH E. CASTILLON.

Name Date Hutchins Oct. 18, 1949 OTHER REFERENCES Clark: J. Biol. Chem.'76, pp. 229-234, (1928).

Murty: Chemical Abstracts 40, p. 3219 (1946).

Winton: The Structure and Composition of Foods, vol. 1, pp. 566-569,Wiley, N. Y., 1932.

Number

1. A PROCESS OF PREPARING A WATER-SOLUBLE, TOXIC, GOSSYPOL PRODUCTCOMPRISING DISSOLVING GOSSYPOL AND PROTEIN IN DILUTE AQUEOUS ALKALI, ANDNEUTRALIZING WITH ACID.